There are almost 400 arthropod-borne viruses, many of which cause serious human disease, fall into 5 major families. Sindbis virus (SV) in the family Togaviridae, genus alphavirus, is the subject of this proposal. By studying the comparative aspects of viral replication in Ae. albopictus mosquito cells and vertebrate cells, we have been able to isolate mutants of SV with novel phenotypes: 1) SVLM21, which is able to grow in mosquito cells which have been deprived of methionine, and have low levels of ado met, 2) SVmpa, which can grow in mosquito cells which have been treated with mycophenolic acid, and have low levels of GTP, 3) Svcp which shows increased cytopathogenicity for mosquito cells, 4) SVcl35 and SVc,158 which are host-restricted in mosquito cells and 5) SVAP15/21 which is restricted in vertebrate cells. Making use of recombinant DNA technology and the "infectious" Toto plasmids which contain a CDNA copy of the Sindbis virus RNA, we are mapping the mutations in these viruses and determining the nucleotide changes responsible for their phenotypes; this approach is also making it possible to associate specific biochemical functions with various of the SV nonstructural (ns) proteins. Thus, most recently we have been able, using SVLM, CDNA, to associate the viral RNA methyltransferase with the nonstructural protein, nspl, and by nucleic acid sequencing to identify two amino acid changes in the SVLM, nspl. Since we wish eventually to study the structure-function relationships of nspl we are developing strategies for the construction of expression vectors which will enable us to produce large amounts of both the standard and mutant nspl. In a similar fashion we shall use SVmpa to identify the protein associated with the RNA capping activity (RNA guanylyltransferase). The significance of these studies lies in the fact that until now there has been little rigorous evidence which makes it possible to associate a specific function with any of the four SV ns proteins. Since SVLM2l and SVMPA, are the only mutants of SV which have both 1) a change in an ns protein and 2) a phenotype which points to an alteration in a specific biochemical function, they will be invaluable for associating at least two functions with specific ns proteins. What we learn from these studies concerning the SV genome and its replication strategy may ultimately lead to novel strategies for the control of medically important viruses, which are transmitted by arthropod vectors.